The global impact of rickettsial infections is illustrated by the resurgence of long-known pathogens, as well as the emergence of newly recognized pathogens. Infections with Rickettsia rickettsii (Rocky Mountain Spotted Fever) continue with severe consequences in South and Central America. The resurgence of R. conorii (Mediterranean Spotted Fever) in Europe, the Middle East and Africa, as well as a recent worldwide rise in murine typhus (R. typhi), highlights the threats of rickettsial diseases. Despite the public health importance of pathogenic Rickettsia spp.,, our limited knowledge of ricketsial biology has been an impediment to progress towards more effective intervention modalities. Our phylogenomics analyses have highlighted considerable variation across Rickettsia genomes, providing a framework to link particular genotypes with their associated disease phenotypes. For several bona fide secretory proteins that have been characterized in universal rickettsial processes (i.e., host cell adhesion, invasion and intracellular growth and survival), a patchy genomic distribution indicates that the mechanisms underpinning these processes are inherently different across rickettsial groups. For instance, our recent work on R. typhi (Typhus Group) identified a novel invasin, RalF, which interacts with host Arf6 in a process dependent on host phosphoinositide PIP2. Curiously, RalF genes are absent from species of Spotted Fever Group (SFG). Conversely, two well-characterized surface proteins (Sca0 and Sca2) of SFG pathogens are either absent (Sca0) or highly divergent (Sca2) in non-SFG rickettsial species. Thus, mechanisms of Rickettsia host cell invasion are more complex than previously appreciated, necessitating the need to employ a comparative approach for investigating the factors underpinning pathogenesis. Under this proposal, our work will focus on identifying the mammalian and invertebrate host cell targets of Sca3 and divergent Sca2 (d-Sca2) proteins from non-SFG species (Aim 1). Additionally, we will investigate the manner by which non- SFG species trigger phosphoinositide (PIP) metabolism to facilitate membrane ruffling and rickettsial endocytosis, with identified host proteins and PIPs present on the early endosome further explored as docking sites for rickettsial phospholipases that mediate phagosome escape (Aim 2). The successful outcome of this work will provide important clues on how divergent Rickettsia species utilize different molecules to achieve the universal rickettsial process of host cytoplasmic infection via induction of phagocytosis. We anticipate this knowledge to yield disease-specific therapeutic approaches to combat fatal rickettsioses.